Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Sarit Mizrachy-Schwartz,Shoshana Klein,Nataly Kravchenko-Balasha,Alexander Levitzki,Hannah Ben-Bassat,Mikhail Blagosklonny

doi:10.1371/journal.pone.0000628

Sarit Mizrachy-Schwartz, Shoshana Klein + Show 4 more

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https://doi.org/10.1371/journal.pone.0000628

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Abstract

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation.

Highlights

The evolution of cancer is a complex, multi-step process characterized by an altered array of signal transduction networks and metabolic pathways [1]
Cap-mediated protein synthesis is the dominant mechanism for generating proteins, the proteins we demonstrated to be highly expressed in L and BP cells – namely, c-Myc, X-linked inhibitor of apoptosis (XIAP), hypoxia-inducible factor 1a (Hif-1a) and vascular endothelial growth factor (VEGF) – can be translated by both cap and IRESdependent mechanisms [9,10,11,12]
In order to follow the biochemical changes that occur when cells are transformed, we established an in-vitro cell system where we have a continuum of cells, from normal human keratinocytes, through consecutive passages of human papillomavirus 16 (HPV16) immortalized cells, all the way to extensively passaged, benzo[a]pyrene treated cells that form colonies in soft agar

Summary

Introduction

The evolution of cancer is a complex, multi-step process characterized by an altered array of signal transduction networks and metabolic pathways [1]. Our analysis includes cells from early passage (E), from late passage (L) and from late passage cells that underwent treatment with benzo[a]pyrene (a carcinogen that is present in cigarette smoke) to generate BP cells, which form colonies in soft agar. In validation of this model, we have shown a striking convergence of gene expression between our transformed cells and the data of Santin et al [6], who investigated changes in expression in cervical carcinoma (Kravchenko-Balasha et al, submitted). Our model enables us to perform molecular analysis of HPV16-transformed keratinocytes at various stages, which represent steps in the transformation process, in order to try to build a global picture of the evolution of cellular transformation

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